All of the previously developed immuno assays in common use rely on a coating of a protein on a solid phase base, in order to react with immune complexes in the specimen of serum, spinal fluid, plasma, saliva, semen, tears, urine or the like. The presence of such immune complexes in such a specimen is indicative that a patient, from whom such a specimen was taken, has a condition such as rheumatoid arthritis, tumor, hepatitis, viral infection or the like. The ELISA test, described more particularly hereinafter, involves coating a solid phase base with a protein, washing with specified solutions, and placing the serum or other sample to be tested on the protein, to permit the protein to adsorb the immune complexes in the specimen. An enzyme labeled anti-species immunoglobulin, such as anti-human IgG coupled with an enzyme such as a phosphatase, is added and permitted to stand for a desired period of time, followed by washing preferably a number of times with a predetermined solution. Then a substrate is added, which will react with the enzyme then attached to the protein and normally change color, dependent on the amount of immune complex which has been bound. With a p-nitro phenyl phosphate, the color produced is yellow, but with other substrates, the color may be different. After a period of time, such as 30 minutes or more, the color change may be observed visually for a generally qualitative determination, although through the use of a spectrophotometer, a more accurate reading may be obtained than could be obtained visually.
Other in vitro assays for the detection of immune complexes include the Raji cell radio-immuno assay which measures (or quantitates) the amount of immune complexes through the use of a radioactive material such as an anti-IgG-I.sup.125 labeled antiserum. After washing off the excess radiolabeled antiserum, the amount of immune complexes reactive with the radioactive material may be ascertained through use of a scintillation counter. The Raji cell radioimmuno assay test is described in In Vitro Methods in Cell Mediated and Tumor Immunity Section D, chapter 52 (Academic Press 1976, edited by Bloom, B. and J. David).
Previously developed isotype specificity tests, for the class or subclass of the antibody component of a circulating immune complex, utilizing a protein for initial reaction with a specimen, have been carried out but have not utilized a monomixture or an admixture of monoclonal or polyclonal anti-antibodies with a single or multiple immunoglobulin isotype, such as IgA, IgD, IgE, IgG, IgG4 or IgM. Determination of the antibody subclass sheds light on both the possible origin and the prognosis of the disease. However, no previous antigen specific assay, as to aid in the identification of circulating immune complex related diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and the like, are known.
Since each of the previous tests require protein for initial reaction with a specimen, it is apparent that a much less expensive material to form a layer on a solid phase base would reduce quite materially the cost of the material used in the immunoassay and thus reduce the ultimate cost. In addition, the use of radioactive material ordinarily requires a special permit, and, more particularly, numerous precautions in handling and use. In addition, the radioactive material is quite expensive and hazardous.
One of the objects of this invention is to provide a novel method of performing an immunoassay of a specimen of body fluid or the like to determine the identity of an antigen of a circulating immune complex and the quantity of that complex; and to provide a novel method of performing an immunoassay of a specimen of a body fluid or the like to determine the class or subclass of an antibody of a circulating immune complex and the quantity of that complex; and to provide such methods which involve the use of a considerably less expensive material than the protein required for previous types of assays; and to provide such methods which will produce reliable and reproducible results; and to provide such methods which can be carried out with more ease and in a more simple manner than in similar tests; and to provide an article which is readily produced in quantity and which will reduce considerably the time involved in the actual assay; and to provide such an article which can be made so that it can be stored for long periods of time; and to provide a method of making such an article, by which the article can be made effectively and efficiently; and to provide such a method which can be carried out easily and effectively and at a significantly lower cost.